The long-term goal of this project is to better understand inflammatory mechanisms of vascular injury and repair and to develop new therapeutic strategies to treat patients with vascular diseases. Abundant evidence indicates that the CD40-CD40 ligand (CD40L) pathway plays a pivotal role in immune and inflammatory responses. Elevated levels of CD40L in the circulation have been associated with hypercholesterolemia, diabetes, acute coronary syndromes, and an increased probability of restenosis. To date there is no direct experimental evidence addressing if and how the CD40L pathway contributes to vascular injury and repair process. The results of our preliminary studies indicate multiple beneficial effects of CD40L blockade on the vascular injury response in animals on an atherogenic background, which is contrary to the limited evidence in a non-atherogenic mouse injury model. Based on our preliminary data, the overall hypothesis of this proposal is that circulating CD40L (both soluble CD40L and platelet-associated CD40L) is elevated and contributes to proinflammatory responses and neointimal formation after vascular injury in an atherogenic environment. To test our hypothesis and achieve our long-term goal, we will employ genetic, pharmacologic, and biochemical approaches in well-characterized cellular and animal models in the following three Specific Aims. Aim 1 will establish the role of CD40L in inflammation and neointima formation after arterial injury (carotid wire and FeCl3 injury) in apoE-/-CD40L-/- mice and apoE-/-CD40L+/+ mice vs in mice deficient in CD40L in a non-atherogenic environment. The relative contribution of CD40L expression by bone- marrow derived cells and vascular wall cells will be determined by creation of chimeric animals by bone marrow transplantation. Aim 2 will establish the contribution of soluble CD40L and platelet-associated CD40L in the inflammatory and thrombotic responses to vascular injury in an atherogenic milieu. We will examine platelet-leukocyte activation and interactions as well as tissue factor (TF) expression in apoE-/-CD40L-/- mice and apoE-/-CD40L+/+ mice, and then determine effects of intravenous reconstitution with recombinant sCD40L or CD40L-containing platelets in the mice on platelet-leukocyte leukocyte recruitment and TF expression at sites of arterial injury. Aim 3 will establish the role of CD40 (the receptor for CD40L) in neutrophil activation and inflammatory functions in vitro, and in the inflammatory and thrombotic responses to vascular injury in vivo. We will determine if CD40 is required for CD40L-mediated effects on neutrophils in vitro using CD40- expressing and CD40-deficient neutrophils, and then determine the role of CD40 in leukocyte recruitment and neointima formation after arterial injury in CD40-deficient (CD40-/- and apoE-/-CD40-/-) mice. In addition, we will determine if CD40 is required for CD40L-mediated TF induction in vivo. The results of these studies will provide mechanistic insight into the role of CD40/CD40L signaling in the vascular response to injury on an atherogenic background and may provide valuable information for the development of novel therapeutic strategies to prevent and treat vascular disease in humans. Project Narrative: Clinical restenosis, defined as e 50% loss of the initial lumen size of vessels, remains a major clinical problem in patients after coronary angioplasty and stent placement. This project is designed to better understand the mechanisms of restenosis using the well established mouse model of vascular injury and repair. The results obtained will provide novel insights into the mechanisms of restenosis and promise to provide valuable information for the development of novel therapeutic strategies to prevent and treat vascular disease in humans.